Novel Carboline Derivatives as Potent Antifungal Lead Compounds: Design, Synthesis, and Biological Evaluation

Article abstract of DOI:10.1021/ml400492t

A series of novel antifungal carboline derivatives was designed and synthesized, which showed broad-spectrum antifungal activity. Particularly, compound C38 showed comparable in vitro antifungal activity to fluconazole without toxicity to human embryonic lung cells. It also exhibited good fungicidal activity against both fluconazole-sensitive and -resistant Candida albicans cells and had potent inhibition activity against Candida albicans biofilm formation and hyphal growth. Moreover, C38 showed good synergistic antifungal activity in combination with fluconazole (FLC) against FLC-resistant Candida species. Preliminary mechanism studies revealed that C38 might act by inhibiting the synthesis of fungal cell wall.

Full text of DOI:10.1021/ml400492t

Letter
pubs.acs.org/acsmedchemlett
Novel Carboline Derivatives as Potent Antifungal Lead Compounds:
Design, Synthesis, and Biological Evaluation
Shengzheng Wang,†,∥ Yan Wang,†,∥ Wei Liu,‡ Na Liu,† Yongqiang Zhang,† Guoqiang Dong,† Yang Liu,†
Zhengang Li,† Xiaomeng He,† Zhenyuan Miao,† Jianzhong Yao,† Jian Li,§ Wannian Zhang,*,†
and Chunquan Sheng*,†
†School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
‡School of Medicine, Tongji University, 1239 Siping Road, Shanghai 200092, People’s Republic of China
§School of Pharmacy, East China University of Science and Technology, Shanghai 200237, People’s Republic of China
S
* Supporting Information
ABSTRACT: A series of novel antifungal carboline deriva-
tives was designed and synthesized, which showed broad-
spectrum antifungal activity. Particularly, compound C38
showed comparable in vitro antifungal activity to fluconazole
without toxicity to human embryonic lung cells. It also
exhibited good fungicidal activity against both fluconazole-
sensitive and -resistant Candida albicans cells and had potent
inhibition activity against Candida albicans biofilm formation and hyphal growth. Moreover, C38 showed good synergistic
antifungal activity in combination with fluconazole (FLC) against FLC-resistant Candida species. Preliminary mechanism studies
revealed that C38 might act by inhibiting the synthesis of fungal cell wall.
KEYWORDS: Antifungal lead compounds, carboline derivatives, fungicidal activity
caspofungin, micafungin, and anidulafungin).3,4 Among them,
n recent years, the incidence and associated mortality of
amphotericin B has been used as the gold standard for almost
Iinvasive fungal infections (IFIs) has been increasing
dramatically.1,2 However, effective and safe antifungal agents
all IFIs for more than 40 years, but severe nephrotoxicity has
been associated with this drug.5,6 5-Fluorocytosine is usually
are very limited. Clinically available antifungals (Figure 1) for
used as adjunctive therapy. In combination with amphotericin
IFIs mainly include: polyenes (e.g., amphotericin B),
B, it is effective against a number of mycoses, including some
fluorinated pyrimidines (e.g., 5-fluorocytosine), azoles (e.g.,
caused by the cryptococcus spp., dematiaceous fungi, and some
fluconazole and voriconazole), and echinocandins (e.g.,
Candida spp. The azole antifungals have emerged as front-line
drugs in antifungal chemotherapy.7,8 Among the azoles,
fluconazole plays an important role in treating both superficial
and invasive yeast fungal infections due to its favorable
pharmacokinetics, excellent activity against Candida spp., and
safety profile.7,8 However, it is not effective against invasive
aspergillosis and has suffered severe drug resistance. Echino-
candin antifungals show potent activity against Candida and
Aspergillus spp. but are not active in other common and
emerging pathogens, such as Fusarium, Scedosporium, and
Zygomycetes.9 Moreover, resistance to echinocandins has also
been reported.10 Therefore, there is a need to discover and
develop antifungal agents with novel chemotype and fungal-
specific mode of action.
Continuing our efforts on antifungal drug discovery,11−15 we
performed a cell-based antifungal screen of an in-house library
using the standard NCCLS (National Committee for Clinical
Laboratory Standards) protocol. Compound 1 with a β-
Received: November 30, 2013
Accepted: February 13, 2014
Published: February 13, 2014
Figure 1. Chemical structures of representative antifungal agents for
the treatment of invasive fungal infections and hit compound 1.
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
carboline scaffold was identified as a modest fungal inhibitor,
with an MIC80 value of 32 and 8 μg/mL against Candida
albicans and Cryptococcus neoformans, respectively. Because the
chemical scaffold of compound 1 differs from that of all
reported antifungal agents, it is interesting to investigate its
structure−activity relationships (SARs) and discover the novel
antifungal lead compound.
Table 1. In Vitro Antifungal Activity of Compounds C1−
a
C18 (MIC80, μg·mL−1)
compd C. alb. C. par. C. neo. C. gla. A. fum. T. rub. M. gyp.
1
32
16
64
32
64
64
64
64
64
>64
64
>64
32
16
>64
16
32
>64
64
64
2
8
>64
32
>64
64
16
32
16
16
16
>64
>64
>64
32
>64
32
64
16
8
C1
64
32
16
16
>64
32
32
32
>64
32
64
16
8
16
C2
64
16
32
8
C3
64
16
64
64
Thus, a series of novel carboline derivatives was designed and
synthesized (Schemes 1 and 2). The in vitro antifungal
C4
>64
>64
>64
>64
>64
>64
>64
32
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
16
>64
>64
32
C5
a
C6
32
Scheme 1. Chemical Synthesis of Compounds C1−C18
C7
32
>64
>64
>64
>64
>64
8
C8
>64
64
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
FLC
>64
32
32
16
>64
64
64
8
64
>64
>64
32
64
64
16
8
16
>64
8
32
8
>64
>64
32
>64
64
16
16
16
2
>64
>64
>64
>64
>64
>64
>64
4
16
32
16
1
>64
0.5
16
0.5
a
a
Abbreviations and strain numbers: C. alb., Candida albicans
Reagents and conditions: (a) K2CO3, EtOH, reflux, 2 h; (b) 1,4-
(SC5314); C. par., Candida parapsilosis (ATCC 22019); C. neo.,
Cryptococcus neoformans (32609); C. gla., Candida glabrata (537); A.
fum., Aspergillus fumigatus (07544); T. rub., Trichophyton rubrum
(Cmccftla); M. gyp., Microsporum gypseum (Cmccfmza); FLC,
fluconazole.
dioxane, conc. H2SO4, 80 °C, 3 h; (c) base (NaH or KOH), solvent
(DMF or DMSO), rt or 45 °C.
a
Scheme 2. Chemical Synthesis of Compounds C19−C42
of C38 against WI38 cell line (human embryonic lung cell) was
determined using the standard MTT method. The IC50 value of
C38 was larger than 40 μg/mL, indicating that it had selective
antifungal activity.
Time-kill testing assay was used to test whether this new type
of antifungal compound had fungicidal activity. The blank
control and FLC at 32 μg/mL were used as controls. As shown
in Figure 2A, compound 1 showed no significant difference
compared with FLC against C. albicans SC5314 (FLC sensitive,
MIC80 = 0.5 μg/mL), indicating that it may only had fungistatic
activity, which was similar to that of FLC. The fungicidal
activity of C38 was also investigated at the concentration range
of 2−32 μg/mL. As shown in Figure 2B, C38 at 2 μg/mL
showed no significant difference compared with FLC at 32 μg/
mL against the C. albicans SC5314 strain. Interestingly, C38
showed fungicidal activity at the concentration of 4 μg/mL.
When the concentration was increased to 8 and 16 μg/mL, the
fungicidal activity was enhanced. To our delight, under the
treatment of C38 at 32 μg/mL for 24 h, density of fungi cells
was reduced to zero, whereas FLC had no significant effect at
the same concentration. The results confirmed that carboline
derivative C38 had excellent fungicidal activity against FLC-
sensitive C. albicans in a dose-dependent manner.
a
Reagents and conditions: (a) K2CO3, CH2Cl2, rt, 6 h; (b) EtOH,
reflux, 2 h; (c) K2CO3, EtOH, reflux, 4 h; (d) DMSO, KOH, rt, 12 h;
(e) EtOH, Claisen hydrolysate, reflux, 3 h.
Importantly, fungal resistance caused by broad use of azoles
is becoming a serious problem in recent years.16 It is highly
desirable to find a new type of inhibitors that could be effective
against azole-resistant fungal cells. C. albicans 103 (FLC
resistant, MIC80 > 64 μg/mL) was used to test whether C38
could be effective against FLC-resistant fungi. As shown in
Figure 2C, C38 obviously inhibited the growth of C. albicans
103 at the concentrations of 4 and 8 μg/mL. When the
concentration was increased to 16 and 32 μg/mL, C. albicans
activities are summarized in Tables 1 and 2. Most of the target
compounds showed moderate to good inhibitory activity with
broad spectrum. Among them, compound C38 showed potent
inhibitory activity against all the tested fungal pathogens
(MIC80 range: 1 to 4 μg/mL). Its activity against C. albicans
(MIC80 = 2 μg/mL), C. neoformans (MIC80 = 1 μg/mL), M.
gypseum (MIC80 = 1 μg/mL), and C. krusei (MIC80 = 4 μg/mL)
was comparable or superior to FLC. Moreover, the cytotoxicity
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An important reason for the severe drug resistance is the
strong ability of Candida cells to form biofilms.17,18 More over,
biofilms could shield Candida cells from attack by the immune
system and also block antifungal agents from reaching infected
cells.19,20 The ability of C38 to inhibit biofilm formation of C.
albicans SC5314 (FLC sensitive) and C. albicans 103 (FLC
resistant) was investigated. FLC at 64 μg/mL and the blank
control were used as controls. For the strains of C. albicans
SC5314 and C. albicans 103 (Figure 3), the fungi cells treated
Table 2. In Vitro Antifungal Activity of Compounds C19−
a
C42 (MIC80, μg·mL−1)
compd C. neo. C. alb. C. par. C. tro. C. krusei T. rub. M. gyp.
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
C31
C32
C33
C34
C35
C36
C37
C38
C39
C40
C41
C42
FLC
32
8
8
16
16
16
16
4
64
16
16
4
16
4
32
8
16
8
8
16
16
4
16
16
4
4
16
32
8
8
4
8
4
4
4
4
8
16
4
16
16
64
16
64
>64
16
16
4
4
16
8
16
4
4
4
4
16
16
8
4
16
16
>64
>64
64
64
4
16
4
4
4
16
8
16
8
16
4
16
16
4
8
8
8
4
16
4
4
8
2
8
4
8
2
8
8
16
16
4
4
4
Figure 3. Effects of different concentrations of compound C38 on
biofilm formation. C. albicans SC5314 (A) and C. albicans 103 (B)
were used in this assay. Cells were treated with compound C38 (2−64
μg/mL) or FLC (64 μg/mL). Biofilm formation (as measured by XTT
reduction) is expressed as a percentage of that of blank control. Results
8
8
4
16
16
64
64
16
4
4
4
4
8
4
2
4
16
4
32
4
64
16
4
64
16
4
32
4
16
4
4
2
4
2
represent the mean
standard deviations for three independent
1
2
4
4
2
1
experiments. Statistical significance among the groups was determined
by the analyses of variance (ANOVA). For comparison between two
groups, the Student t test was used. A p-value of less than 0.05 was
considered significant. *p < 0.05 compared to the control group; **p
< 0.01 compared to the control group.
32
4
32
8
4
16
16
16
4
4
32
4
16
4
4
4
8
4
64
64
2
4
2
1
4
8
4
8
4
2
0.5
1
4
4
1
a
Abbreviations and strain numbers: C. alb., Candida albicans
with 64 μg/mL of FLC showed no inhibitory effect of biofilm
formation and had no significant difference compared with the
blank control, which was consistent with the previous
reports.21−23 Interestingly, C38 showed a potent inhibitory
effect on biofilm formation. Moreover, the inhibitory effect
appeared to be dose-dependent. As shown in Figure 3A, C38
could inhibit >50% of biofilm formation at the concentration of
4 μg/mL. When the concentration was increased to 64 μg/mL,
biofilm formation was significantly inhibited (>80%). More
importantly, C38 also showed potent inhibitory effect on
biofilm formation of the FLC-resistant C. albicans 103 strain
(Figure 3B). It inhibited biofilm formation by >75% at the
concentration of 32 μg/mL. The biofilm inhibition assay
confirmed that C38 showed dose-related inhibitory activity on
biofilm formation and that it was effective against both FLC-
sensitive and FLC-resistant fungi.
(SC5314); C. par., Candida parapsilosis (ATCC 22019); C. neo.,
Cryptococcus neoformans (32609); C. tro., Candida tropicalis; C. krusei,
Candida krusei (0710397); T. rub., Trichophyton rubrum (Cmccftla);
M. gyp., Microsporum gypseum (Cmccfmza); FLC, fluconazole.
A striking feature of C. albicans biology is its ability to grow
in a variety of morphological forms, such as yeast,
pseudohyphae, and hyphae.24 In particular, the hyphae form
could promote fungi cells to penetrate human epithelial and
endothelial cells during the early stages of infection and cause
damage.25,26 Moreover, hyphae is essential for pathogenicity by
forming biofilms,27 which are highly resistant to standard
antifungal treatments. Therefore, the inhibitory ability of C38
on the growth of C. albicans hyphae was determined (Figure 4).
In the blank control group (Figure 4A), most of the C. albicans
cells showed elongated hyphae forms. After treated with FLC at
8 μg/mL (Figure 4B), the hyphal growth was not inhibited, and
the morphology was similar to that of the blank control. The
result was consistent with the fact that FLC was not effective in
C. albicans hyphal growth.28,29 After treated with C38 at 2 μg/
mL (Figure 4C), most of the C. albicans cells appeared as
pseudohyphae form, and the cells were much shorter than the
control group. More interestingly, only unicellular budding
Figure 2. Time-kill curves of C. albicans obtained by using an initial
inoculums of 105 CFU/mL. CFUs/mL were determined after 0, 12,
24, and 48 h of incubation. (A) C. albicans SC5314 (FLC-sensitive,
MIC80 = 0.5 μg/mL) was treated/untreated with FLC (32 μg/mL) or
lead compound 1 (32 μg/mL). (B) C. albicans SC5314 was treated/
untreated with FLC (32 μg/mL) or different concentrations of
compound C38. (C) C. albicans 103 (FLC-resistant, MIC80 > 64 μg/
mL) was treated/untreated with FLC (32 μg/mL) or different
concentrations of compound C38.
cells were decreased to a larger extent. The results confirmed
that compound C38 had potent fungicidal activity against FLC-
resistant C. albicans.
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Figure 5. Transmission electron micrographs of C. albicans cells under
different conditions. The organelles indicated by the arrows are as
follows: 1, cell wall; 2, cell membrane; 3, the outer sphere of cell wall;
4, the middle sphere of cell wall; 5, vacuole; 6, reduced cytoplasmic
density. The normal C. albicans cell (A) shows a uniform central
density and a regularly outlined cell wall. The cell treated with FLC at
8 μg/mL for 8 h (B) shows damaged cell membrane and abnormal
shape. Picture C shows the cell treated with compound C38 at 8 μg/
mL for 8 h. It shows that the outer sphere of cell wall has become
thinner and that the middle sphere has become thicker. Moreover, the
reduced cytoplasmic density, necrosis, and some abnormal shape of
vacuoles could also be observed.
Figure 4. Effects of different concentrations of compound C38 or FLC
on C. albicans SC5314 hyphal growth. The fungi cells were incubated
in RPMI 1640 liquid medium at 37 °C for 16 h in the absence or
presence of different concentrations of compound C38 (2 μg/mL to 8
μg/mL) or FLC (8 μg/mL). The hyphal development was viewed by
light microscopy at ×400 and photographed.
yeast form was observed after exposure with C38 at 4 μg/mL or
higher concentration (Figures 4D−F). In this assay, C38
showed potent inhibitory effect on the C. albicans hyphal
growth, whereas FLC was ineffective.
The in vitro synergistic antifungal activity of compound C38
in combination with FLC was also conducted (Table 3). Four
sphere of the cell wall became thinner, and the middle sphere
became thicker. No obvious damage was observed on cell
membrane. Some abnormal shapes of the vacuoles were also
observed. In addition, reduced cytoplasmic density and necrosis
could be observed. The TEM results indicated that C38 caused
significant damage on C. albicans cells. Obvious cell wall change
suggests that this new type of antifungal compound may have
an effect on some synthetic routes of fungal cell wall.
Table 3. In Vitro Synergistic Antifungal Activities of
Compound C38 and FLC (MIC80, μg·mL−1)
GC-MS method was used to analyze the change of sterol
composition in C. albicans cells, which could reflect the effect
on C. albicans membrane. The assay has been successfully used
in studying the mechanism of fungal resistance31,32 and the
influence of inhibitors on sterol biosynthesis pathway.33
Cholesterol was added as internal standard and the results
were shown in Table 4. In the blank control group, ergosterol
MIC80 in
combina-
MIC80 alone
tion
clinical
isolates
mode of
a
FLC
C38 FLC C38 FICI
interaction
103
>1024
>1024
>1024
>1024
4
4
4
4
2
2
2
2
1
1
1
1
0.252
0.252
0.252
0.252
syn
syn
syn
syn
100
07109
805
Table 4. Relative Sterol Composition of C. albicans SC5314
a
after 24 h of Incubation
Synergy and antagonism were defined by FICI ≤ 0.5 and >4,
respectively. Indifferent was defined by 0.5 < FICI ≤ 4.
a
% of total sterols (Candida albicans SC5314)
b
c
d
sterol
control
C38
FLC
FLC-resistant C. albicans spp. (MIC80 > 1024 μg/mL) were
used in this testing. To our delight, C38 alone showed good
inhibitory activities against all tested Candida species with MIC80
values of 4 μg/mL. More interesting, C38 showed good
synergistic activities in combination with FLC. The fractional
inhibitory concentration index (FICI) value of C38 was 0.252,
indicating C38 exhibited good synergistic activities against
tested Candida species in combination with FLC. In this assay,
C38 not only showed good antifungal activities against FLC-
resistant Candida species but also showed potent synergistic
activities in combination with FLC.
In order to reveal the antifungal mechanism of compound
C38, its influence on the morphology of C. albicans was
monitored by transmission electron microscopy (TEM). The
normal C. albicans cells and cells treated with FLC at 8 μg/mL
for 8 h were used as controls. As shown in Figure 5A, the
normal C. albicans cell has a uniform central density and an
intact cell wall. For the cells treated with FLC (Figure 5B), the
cell membrane was damaged obviously, and the shape of the
cells became abnormal. These alterations caused by FLC were
consistent with the reported results that FLC acts on fungal cell
membrane.30 After exposure to C38 (Figure 5C), the outer
ergosterol
obtusifoliol
lanosterol
eburicol
90.0
0.0
1.5
0.0
8.5
40.2
3.7
0.6
6.5
5.9
12.2
12.4
31.5
82.2
4.8
unidentified
a
b
d
Sterol proportions varied by less than 10% in three experiments.
Control (no drug). Treated with compound C38 at 8 μg/mL.
Treated with FLC at 8 μg/mL. The data was the average of three
independent experiments.
c
and lanosterol comprised 90.0% and 1.5% of the total sterol
fraction, respectively. Other 14-methylated sterols such as
obtusifoliol and eburicol were not observed. When the cells
were treated with FLC at 8 μg/mL for 24 h, the content of
eburicol was increased up to 82.2% of the total sterol fraction.
Other 14-methylated sterols (lanosterol and obtusifoliol) also
increased obviously. In contrast, ergosterol content was reduced
to lower than 1% in FLC treated group. These alternations
were caused by competitive inhibition of CYP51 by FLC and
consistent with the reported results.31,32 Interestingly, treat-
ment with C38 also resulted in a significant reduction of
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ACS Medicinal Chemistry Letters
Letter
ergosterol content (reduced to 40%) and an obvious
accumulation of eburicol and lanosterol precursors (Table 4).
However, the sterol composition change was not as obvious as
FLC induced effects. The level of ergosterol (about 40% of the
total sterol fraction) may not induce obvious change of fungal
membrane structure, which was consistent with the results that
no obvious damage was observed on cell membrane in the
TEM testing. The GC-MS results infer that this new type of
inhibitor might have a different mode of action with FLC.
Cytochrome P450 (CYP) enzymes play an important role in
the metabolic clearance of the vast majority of prescribed drugs.
The assessment of CYP enzymes inhibition or inactivation
remains an important role in overall drugs’ safety assessment.
Clinical drug−drug interactions (DDI) caused by inhibiting
these CYP enzymes can result in dangerous side effects, caused
by reduced clearance/increased exposure of a second drug.34
Thus, the DDI potential for compound C38 was investigated in
vitro using four major human CYP isoforms (2C19, 2C9, 3A4-
M, and 3A4-T). As shown in Table 5, compound C38 had weak
AUTHOR INFORMATION
Corresponding Authors
*(C.S.) Phone/Fax: 86-21-81871239. E-mail: shengcq@
hotmail.com.
*(W.Z.) Phone/Fax: 86-21-81871243. E-mail: zhangwnk@
hotmail.com.
■
Author Contributions
∥S.W. and Y.W. contributed equally to this work.
Funding
This work was supported in part by National Natural Science
Foundation of China (grants 81222044 and 81273558), Key
Project of Science and Technology of Shanghai (grant
13431900301), the 863 Hi-Tech Program of China (grant
2012AA020302), the National Basic Research Program of
China (grant 2014CB541800), Shanghai Rising-Star Program
(grant 12QH1402600), and Shanghai Municipal Health Bureau
(grant XYQ2011038) for financial support.
Notes
The authors declare no competing financial interest.
Table 5. In Vitro CYP Inhibition Assessment of Compound
C38
ABBREVIATIONS
■
IFI, Invasive fungal infection; AmB, amphotericin B; FLC,
fluconazole; SAR, structure−activity relationship; FICI, frac-
tional inhibitory concentration index; CYP, cytochrome P450;
DDI, drug−drug interactions; TEM, transmission electron
microscopy
% inhibition
IC50
potential
a
compd isoenzyme 25 μM 2.5 μM 0.25 μM (μM)
inhibition
C38
C38
C38
C38
2C19
2C9
33
5
4
>25
>25
50
low
low
low
low
36
8
−2
1
3A4-M
3A4-T
−35
50
−10
10
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In summary, a series of novel antifungal carboline derivatives
was designed and synthesized. The most active compound C38
showed several advantages as a promising antifungal lead: (1) it
showed broad-spectrum antifungal activity, comparable to FLC;
(2) it revealed potent fungicidal activity against both FLC-
sensitive and FLC-resistant C. albicans cells, whereas FLC only
showed fungistatic activity; (3) it was a good inhibitor of C.
albicans biofilm formation and hyphal growth, suggesting its
potential to overcome FLC-related drug resistance; (4) it
showed good synergistic antifungal activities in combination
with FLC against FLC-resistant Candida species; (5) it might
have a new mode of action resulting from inhibiting the
synthetic route of fungal cell wall; (6) Preliminary CYP
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against tested human CYP isoforms, indicating it had low
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